Background: The efficiency of CD4/CCR5 mediated HIV-1 entry has important implications for pathogenesis and transmission. The HIV-1 receptor affinity profiling (Affinofile) system analyzes and quantifies the infectivity of HIV-1 envelopes (Envs) across a spectrum of CD4/CCR5 expression levels and distills these data into a set of Affinofile metrics. The Affinofile system has shed light on how differential CD4/CCR5 usage efficiencies contributes to an array of Env phenotypes associated with cellular tropism, viral pathogenesis, and CCR5 inhibitor resistance. To facilitate more rapid, convenient, and robust analysis of HIV-1 entry phenotypes, we engineered a reporter Affinofile system containing a Tat- and Rev-dependent Gaussia luciferase-eGFP-Reporter (GGR) that is compatible with the use of pseudotyped or replication competent viruses with or without a virally encoded reporter gene. This GGR Affinofile system enabled a higher throughput characterization of CD4/CCR5 usage efficiencies associated with differential Env phenotypes.

Results: We first validated our GGR Affinofile system on isogenic JR-CSF Env mutants that differ in their affinity for CD4 and/or CCR5. We established that their GGR Affinofile metrics reflected their differential entry phenotypes on primary PBMCs and CD4+ T-cell subsets. We then applied GGR Affinofile profiling to reveal distinct entry phenotypes associated with transmission, subtype specificity, and resistance to broadly neutralizing antibodies (BNAbs). First, we profiled a panel of reference subtype B transmitted/founder (T/F) and chronic Envs (n = 12) by analyzing the infectivity of each Env across 25 distinct combinations of CD4/CCR5 expression levels. Affinofile metrics revealed that at low CCR5 levels, our panel of subtype B T/F Envs was more dependent on high levels of CD4 for HIV-1 entry compared to chronic Envs. Next, we analyzed a reference panel of 28 acute/early subtype A-D Envs, and noted that subtype C Envs could be distinguished from the other subtypes based on their infectivity profiles and relevant Affinofile metrics. Lastly, mutations known to confer resistance to VRC01 or PG6/PG19 BNAbs, when engineered into subtypes A-D Envs, resulted in significantly decreased CD4/CCR5 usage efficiency.

Figure 5: Sensitivity vector metrics reveal differences in CD4/CCR5 usage efficiencies between Transmitter/Founder (T/F) and chronic envelopes. Normalized infection data using T/F and chronic Env clones were analyzed using VERSA. (A) Vector angle, (θ), (B) mean infectivity (M), and (C) vector amplitude (Δ) values were obtained for each Env clone. Each Env was profiled twice, in triplicate, across 25 combinations of CD4/CCR5 expression. Average metrics of 6 individuals from each group (T/F or chronic, N=12) are shown, each group consisting of 900 data points. The median value of each metric for the T/F and chronic Env cohorts is marked by a line. p values were generated by the non- parametric unpaired t test (***p = 0.0003; *p = 0.05). (D and E) The normalized infectivity for the chronic (blue line) and T/F envelopes (red line) are averaged, and compared as a group at (D) low and (E) high levels of CCR5 expression, across varying levels of CD4 as indicated. (F) Wedge plot of the average angle and amplitude (+/- S.D.) obtained for T/F (dark grey) versus chronic envelopes (light grey). (G) The infectivity profile of individual T/F and chronic Envs (from Additional file5: Figure S3) were averaged to form their respective group profile. 2-D contour plots representing the averaged infectivity profiles of T/F and chronic envelopes are shown. (H) T/F Envs and macrophage tropic (YU2, ADA) and non-macrophage tropic (JRCSF) R5 Envs were used to produce Env pseudotyped luciferase reporter viruses, which were subsequently titrated on JC53 cells. Monocyte derived macrophages were inoculated with equivalent infectious units of each reporter virus, and luciferase activity measured in cell lysates at 72hrs post infection. Results of infection in 3 independent donors are shown. Results are means of triplicate wells, and error bars represent standard deviations.

Mentions:
An accumulating body of evidence indicates that the majority of primary infections are established by a single viral clone[47-49]. To discern whether relevant differences in entry efficiencies exist between T/F and chronic Envs, we used the GGR Affinofile system to examine the infectivity of T/F Envs (isolated from acutely infected Feinberg stage II or III patients)[50], and compared their Affinofile GGR metrics (θ, ∆, M) with those from a standard panel of chronic Envs. The specific clones used are indicated in [see Additional file2: Table S1]. The infectivity of each T/F and chronic Env was examined at 25 distinct CD4/CCR5 expression levels [see Additional file3: Figure S2A-B], and their infectivity metrics (Figure 5A-C) were obtained via VERSA as described in methods.

Figure 5: Sensitivity vector metrics reveal differences in CD4/CCR5 usage efficiencies between Transmitter/Founder (T/F) and chronic envelopes. Normalized infection data using T/F and chronic Env clones were analyzed using VERSA. (A) Vector angle, (θ), (B) mean infectivity (M), and (C) vector amplitude (Δ) values were obtained for each Env clone. Each Env was profiled twice, in triplicate, across 25 combinations of CD4/CCR5 expression. Average metrics of 6 individuals from each group (T/F or chronic, N=12) are shown, each group consisting of 900 data points. The median value of each metric for the T/F and chronic Env cohorts is marked by a line. p values were generated by the non- parametric unpaired t test (***p = 0.0003; *p = 0.05). (D and E) The normalized infectivity for the chronic (blue line) and T/F envelopes (red line) are averaged, and compared as a group at (D) low and (E) high levels of CCR5 expression, across varying levels of CD4 as indicated. (F) Wedge plot of the average angle and amplitude (+/- S.D.) obtained for T/F (dark grey) versus chronic envelopes (light grey). (G) The infectivity profile of individual T/F and chronic Envs (from Additional file5: Figure S3) were averaged to form their respective group profile. 2-D contour plots representing the averaged infectivity profiles of T/F and chronic envelopes are shown. (H) T/F Envs and macrophage tropic (YU2, ADA) and non-macrophage tropic (JRCSF) R5 Envs were used to produce Env pseudotyped luciferase reporter viruses, which were subsequently titrated on JC53 cells. Monocyte derived macrophages were inoculated with equivalent infectious units of each reporter virus, and luciferase activity measured in cell lysates at 72hrs post infection. Results of infection in 3 independent donors are shown. Results are means of triplicate wells, and error bars represent standard deviations.

Mentions:
An accumulating body of evidence indicates that the majority of primary infections are established by a single viral clone[47-49]. To discern whether relevant differences in entry efficiencies exist between T/F and chronic Envs, we used the GGR Affinofile system to examine the infectivity of T/F Envs (isolated from acutely infected Feinberg stage II or III patients)[50], and compared their Affinofile GGR metrics (θ, ∆, M) with those from a standard panel of chronic Envs. The specific clones used are indicated in [see Additional file2: Table S1]. The infectivity of each T/F and chronic Env was examined at 25 distinct CD4/CCR5 expression levels [see Additional file3: Figure S2A-B], and their infectivity metrics (Figure 5A-C) were obtained via VERSA as described in methods.

Background: The efficiency of CD4/CCR5 mediated HIV-1 entry has important implications for pathogenesis and transmission. The HIV-1 receptor affinity profiling (Affinofile) system analyzes and quantifies the infectivity of HIV-1 envelopes (Envs) across a spectrum of CD4/CCR5 expression levels and distills these data into a set of Affinofile metrics. The Affinofile system has shed light on how differential CD4/CCR5 usage efficiencies contributes to an array of Env phenotypes associated with cellular tropism, viral pathogenesis, and CCR5 inhibitor resistance. To facilitate more rapid, convenient, and robust analysis of HIV-1 entry phenotypes, we engineered a reporter Affinofile system containing a Tat- and Rev-dependent Gaussia luciferase-eGFP-Reporter (GGR) that is compatible with the use of pseudotyped or replication competent viruses with or without a virally encoded reporter gene. This GGR Affinofile system enabled a higher throughput characterization of CD4/CCR5 usage efficiencies associated with differential Env phenotypes.

Results: We first validated our GGR Affinofile system on isogenic JR-CSF Env mutants that differ in their affinity for CD4 and/or CCR5. We established that their GGR Affinofile metrics reflected their differential entry phenotypes on primary PBMCs and CD4+ T-cell subsets. We then applied GGR Affinofile profiling to reveal distinct entry phenotypes associated with transmission, subtype specificity, and resistance to broadly neutralizing antibodies (BNAbs). First, we profiled a panel of reference subtype B transmitted/founder (T/F) and chronic Envs (n = 12) by analyzing the infectivity of each Env across 25 distinct combinations of CD4/CCR5 expression levels. Affinofile metrics revealed that at low CCR5 levels, our panel of subtype B T/F Envs was more dependent on high levels of CD4 for HIV-1 entry compared to chronic Envs. Next, we analyzed a reference panel of 28 acute/early subtype A-D Envs, and noted that subtype C Envs could be distinguished from the other subtypes based on their infectivity profiles and relevant Affinofile metrics. Lastly, mutations known to confer resistance to VRC01 or PG6/PG19 BNAbs, when engineered into subtypes A-D Envs, resulted in significantly decreased CD4/CCR5 usage efficiency.